Phylogenetic Analysis Of Ceratopogonidae In Hainan Island

Ceratopogonidae(Diptera: Nematocera),has a large number of species and diverse diets.Many species are important vectors for some diseases,pollinating insects or potential natural enemy for pest control.In this study,collecting nets and light traps were used to collect the midges in 13 cities and counties of Hainan Island.Species were identified by combining morphological characters and DNA barcodes,25 species belongs to 13 genera were sequenced for 5 genes including CO?,28 S r DNA,CAD,18 S r DNA and AATS1.Finally,the phylogeney of Ceratopogonidae were reconstructed using bayesian method and maximum likelihood method.The main findings are as follows:1.A total of 140 permanent slides of midges were made,and sorted to obtain midges in Hainan Island,4 subfamilies,13 genera and more than 100 individuals.One new species Nilobezzia bamenwana,and 3 new record species of China including Nilobezzia minor,Monohelea orientalis,Alluaudomyia spinosipes,and 4 new record species of Hainan including Alluaudomyia angulata,Alluaudomyia crumena,Stilobezzia inermipes,Alluaudomyia tripartite,were identified and described in this study.2.DNA sequences of species included in this study except for Nilobezzia minor,Culicoides sumatrae and Culicoides peregrinus were sequenced for the first time.After analyzing the gene sequences,the result shows:(1)The base composition of the 5 genes exhibit AT bias,among which the CO? gene is up to 65.4%,which is consistent with the insect mitochondrial gene.(2)Nucleotide variablesites,conservedsites,average genetic distance,identity and divergence among the 5 gene sequences were also analysed,which shows that 28 S r DNA and 18 S r DNA are relatively conservative,while the other 3 genes are much more variable.(3)The scatter plots of the analysis of the saturation sites of the 5gene sequences were made all show a linear relationship between transition and transversion,proving that every gene is still not saturated.3.Phylogeny of Ceratopogonidae were constructed based on single gene respectively and five genes combined using the bayesian method and the maximum likelihood method.the result shows:(1)All phylogenetic trees constructed based on single gene shows a unreliable topology because of the low supporting rate.(2)Both of the bayesian tree and the maximum likelihood tree constructed by the conbined five genes shows all genera and all the four subfamilies including Leptoconopinae,Dasyheleinae,Forcipomyiinae,and Ceratopogoninae,are monophyletic and strongly supported.Among them,Subfamily Leptoconopinae and Dasyheleinae were constituted of single genus,Leptoconop and Dasyhelea separately;Subfamily Forcipomyiinae were constituted of Forcipomyia and Atrichopogon,while Subfamily Ceratopogoninae were constituted of Culicoides and all the predatory genera,and the phylogenetic relationship among the 4subfamilies is:(Leptoconopinae +((Dasyheleinae + Forcipomyiinae)+ Ceratopogoninae))which is consistent with the study based on morphological characters and fossils;In the subfamily Ceratopogoninae,trible Culicoidini,Johannsenomyiin and Palpomyiin are monophyletic with high support value(pp>90%,bp>90%)except for Ceratopogonini is polyphyletic,and the phylogenetic relationship is(Palpomyiini +(Sphaeromiini +Johannsenomyiini)).